Method of producing coenzyme q10 by microorganisms

ABSTRACT

Certain yeasts and bacteria produce a large amount of intracellular coenzyme Q10 which is useful as medicine.

United States Patent 91 Kondo et al.

[ Oct. 30, 1973 METHOD OF PRODUCING COENZYME Q10 BY MICROORGANISMS [75] Inventors: Keji Kondo; Yuzo Yamada, both of Shizuoka-ken; Koji Mitzugi; Shin-Ichiro Otsuka, both of Kanagawa-ken, all of Japan [73] Assignee: Ajinomoto Co., Inc., Tokyo, Japan [22] Filed: Mar. 8, 1971 [21] Appl. N0.: 122,226

[30] Foreign Application Priority Data Mar. 31, 1970 Japan 45/27329 Aug 24, 1970 Japan 45/74012 [52] US. Cl. 195/82 [51] Int. Cl. C12c 11/00 Primary ExaminerA. Louis Monacell Assistant Examiner-R. B. Penland Attorney-Kelman & Berman [5 7] ABSTRACT Certain yeasts and bacteria produce a large amount of intracellular coenzyme Q which is useful as medicine.

3 Claims, No Drawings 1 METHOD OF PRODUCING COENZYME Q10 BY MICROORGANISMS The present invention relates to a method of producing coenzyme Q10.

Coenzyme Q is one of the compounds having the general formula CHaO CHaO

and is the compound in which n is 10.

Coenzyme Q can be widely found in animal and plant tissue, and is known to be an essential compound in a terminal electron transport system. The pharmaceutical and physiological effect of coenzyme Q10 in various diseases has recently been found.

Coenzyme Q has been commercially produced by extracting animal tissues, however, this is very expensive. It is known that coenzyme Q1 is present in the cells of bacteria, such as Pseudomonas denitrificans and Agrobacterium tumefaciens, and moulds, such as Neurospora crassa and Aspergillus fumigatus, in very small amounts.

It has now been found that coenzyme Q, can be produced in large amounts in microbial cells of the genera Rhodotorula, Cryptococcus, Sporobolomyces, Candida, Torulopsis, Rhodosporidium, Trichosporon, Aureobasidium, Tremella, Bullera which are yeasts of the bacterium Alcaligenes, when they are cultured on a nutrient medium, and that the coenzyme Q can be easily recovered from the cells.

Microorganisms able to produce coenzyme Q include Rhodotorula flava lFO ll93 Rhodotorula glutinis [F0 667 Rhodotorula rubra FERM P-632 Rhodotorula peneaur lFO 930 Rhodotorula pallida lFO 715 Rhodotorula marina 1E0 879 Rhodotorula texensis [F0 932 Cryplococcus neoformans FERM P-63l Cryptococcus albidus lFO 610 Cryptococcus laurentii [F0 609 Cryptococcus tereus lFO 727 Cryptococcus Iuteolus IFO 61 l Sporobolomyces roseus lFO l03l Sporobolomyces pararoseus lFO l 103 Sporobolomyces salmonicolor [F0 374 Sporobolomyces gracilis FERM P-633 Candida curvata ATCC 10567 Candida curvala FERM P-633 Candida japonica FERM P-662 Candida bogoriensis FERM P-666 Candida marina FERM P-854 Torulopsis ingeniosa FERM P-665 Torulopsis capsuligenes FERM P-667 Torulopsis aeria FERM P-664 Trichosporon byrdii FERM P-85l Trichosparon cutaneum FERM P-850 Rhodosporidium sphaerocarpum F ERM P-856 Rhodosporidium dibovatum FERM P-857 Aureobasidium pullulans F ERM P-852 TremeIIa fuciformis FERM P-85 3 Bullera alba FERM P-855 Alcaligenes faecalis FERM P-849 The microorganisms which can produce coenzyme Q are cultured on a nutrient medium containing as assimilable carbon source, an assimilable nitrogen source, inorganic salts and organic nutrients. Suitable assimilable carbon sources are carbohydrates, such as glucose or starch hydrolyzate, organic acids, such as acetic acid, fumaric acid, maleic acid or lactic acid, alcohols, such as ethanol or propanol and gaseous or liquid hydrocarbons. Suitable assimilable nitrogen sources are organic or inorganic componds, such as amino acids, polypeptone, soybean protein extracts, soybean whey, urea, ammonium sulfate, ammonium chloride, ammonium phosphate, aqueous and gaseous ammonia. Minor amounts of organic compounds, such as yeast extract, meat extracts, malt extract, corn steep liquor and vitamins should be present.

The microorganisms are cultured at pH 2 to 8, at 20 to 37C. for 10 to 50 hours under aerobic conditions. When the pH of the medium changes sufficiently during the cultivation that the microbial growth is inhibited, the pH is adjusted by adding an acidic or alkaline compound.

Microbial cells grown in the culture broth can be recovered by centrifuging or filtration. As the microbial cells contain a large amount of coenzyme Q the cells can be used as nutrients and medicines. Coenzyme 0 may also be isolated from the cells by conventional methods. For example, the microbial cells may be heated under reflux conditions at to C. for about one hour in a mixture of aliphatic lower alcohol, sodium hydroxide and pyrogallol. To coenzyme Q liberated is extracted by a solvent, such as n-hexane or light oil, and solids are removed by filtration. The solvent fraction is concentrated and purified by means of silicagel.

Coenzyme Q so obtained was identified by paper chromatography, thin layer chromatography, elementary analysis, melting point, infrared and ultraviolet absorption spectra and mass spectrometry.

The amount of coenzyme Q10 produced was determined by Folkers method (Archives of Biochemistry and Biophysics, volume 87, page 298, 1960).

EXAMPLE 1 A culture medium containing 5 g/dl glucose, 0.5 g/dl polypeptone and 0.2 g/dl yeast extract, of pH 6.0 was prepared, and one litre batches of the medium were placed in 5 litre Erlenmeyer flasks and sterilized at l20C for 10 minutes. Each medium was inoculated with 50 ml seed culture of Rhodotorula rubra FERM P-632 which had previously been cultured at 25C for 20 hours on the same medium and cultured at 25C for 30 hours with shaking. Ten litres of the culture broth were centrifuged, the microbial cells obtained were washed with water, and centrifuged again. 125 Grams living cells were suspended in ml water, 240 ml methanol, 12 g pyrogallol, 64 g NaOH and 80 ml water were added to the cell suspension, and the mixture was heated to reflux at 80C for one hour. After cooling, the the heated mixture was centrifuged to remove the cells, 320 ml n-hexane was added to the supernatant, and coenzyme Q10 was extracted. Extraction with nhexane was repeated 3 times, and the n-hexane layers were collected, and washed 3 times with lOO ml water. The extract was dried with 50 g anhydrous sodium sulfate, evaporated to dryness under reduced pressure,

and the residues were dissolved in 40 ml acetone. The acetone was evaporated, and the residue was dissolved in 5 ml acetone and passed through a column packed with 200 ml silicagel. Benzene was passed over the column, fractions which contained carotenoid pigment were eluted in the range of the 380 ml fraction, and 150 ml yellow fractions were obtained after the carotenoid fractions were collected. The yellow fractions were evaporated in a vacuum, and the residue was dissolved in 5 ml ethanol. When cooled for two days in an icebox, the solution yielded yellow crystals which were collected by filtration and recrystallized twice from ethanol, and 2.7 mg yellow crystals of coenzyme Q were obtained.

The Rf value on a paper chromatogram of the crystal was identical that of authentic coenzyme Q10, the crystal melted at 48.5C., a maximum absorption band was found at 275 mu in an ultraviolet absorption spectrum of their ethanol solution and E was 165 at 275 mu. A parent peak (a molecular ion peak) was found at m/l 862 by spectrophotometry of an acetone solution. These properties of the crystals were identical which those of authentic coenzyme Q EXAMPLE 2 Sporobolomyces roseus [F 1031 was cultured on a medium containing g/dl glucose, 1 g/dl polypeptone, l ml/dl soybean protein hydrolyzate and 1 ml/dl corn steep liquor, of pH 6.0 as in Example 1, and 160 g living cells were obtained from litres culture broth. The cells were treated in the same manner as in Example 1, and 3.5 mg pure crystalline coenzyme Om was obtained.

EXAMPLE 3 A culture medium containing l g/dl glucose, 0.2 g/d] yeast extract and 0.3 g/dl polypeptone, of pH 6.0 was prepared, 50 ml batches of the medium were placed in 500 ml shaking flask, and yeasts as indicated in the following Table l were cultured at 26 27C. for 36 hours with shaking. Living cells were collected by centrifugation, and coenzyme O was determined as follows:

20 To 30 g ofliving cells were suspended in 30 ml water. 80 Ml methanol, 3 g pyrogallol, 16 g NaOH and 20 ml water were added to the suspension, and the mixture was heated to reflux on a water bath at 80 to 90C and thereafter left to stand at room temperature for 30 minutes. Coenzyme Ow was extracted with 80 ml n-hexane three times, and the combined n-hexane solution was washed with water and dried with anhydrous sodium sulfate. The n-hexane was evaporated under a reduced pressure, and the residue was dissolved in 10 ml acetone. After filtration, the acetone solution was evaporated in a vacuum, and the residue obtained was dissolved in a small amount of acetone. The solution was spotted on a silica gel thin layer, which was developed with benzene, whereby coenzyme Q was separated from carotenoid compounds. A coenzyme 0,, fraction was cut out, dissolved in acetone, and analyzed by paper chromatography. The acetone solution was spotted on a filter paper which had previously been treated @127 .tulxlsilissa 5 5-54 Gr s M r manu astured by Shin-Etsu Chemical Industry Co., Ltd.)- chloroform solution, and developed by the following solvents, together with authentic samples of coenzyme O8 to 10- Solvent A=ethanolz ethyl acetate: water= 5:3:1

(parts by volume) Solvent B=n-propanol: water= 4:1 (parts by volume) Solvent C=Acetone1 water=5:l (parts by volume) Each sample was also spotted on a filter paper which had been treated with 2.5% (w/v) colorless vaselinetoluene solution, and developed by a solvent D (N,N-dimethylformamide water 97:3, parts by volume).

vention were identical with those of the authentic coenzyme Q10.

The amount of the coenzyme Q10 produced was determined by Folkers method. That is, the acetone extract obtained from the thin layer chromatogram was dissolved in 10 ml ethanol, 4 ml of the ethanol solution were mixed with 1 ml ethylcyanoacetate and 1 ml 0.2NKOH solution, and 8 minutes later the light absorbancy was measured at 625 mp by means of a spectrophotometer. On the other hand, a standard light absorbancy value was determined for a mixture of 4 ml ethanol, and 1 ml ethanol and 1 ml 0.2NKOH solution in the same way as for the tested sample. The light absorbancy of the authentic sample was deducted from that of the tested sample, and the amount of coenzyme Q was determined from a standard curve which had been prepared with authentic coenzyme Q10.

TABLE 1 Strain Amount of coenzyme Q,

produced mg/g of dried cells Rlxodomrulu flava IF 0 1 193 0.25 glulinus lFO 667 0.21 peneaus [F0 930 0.22

pallida lFO 715 0.24

marina lFO 879 0.21

ruhra FERM P-632 0.32

texensis IFO 932 0.19 Cryplocuccus neoformans FERM P63! 035 nlbidus lFO 610 0.23 Laurenlii [F0 609 0.20

lereus lFO 727 0.19

luteulus ER) 61 1 0.15 Spombolnmyces reseus lFO 1031 0.36 pararoseus [F0 1 103 0.34 salmonitulur lFO 374 0.35

gracilis FERM P-633 0.31

EXAMPLE 4 The cells were treated as in Example 1, but coenzyme Q was extracted from the silica gel with isooctane instead of benzene, and 18.6 mg pure, yellow, crystalline coenzyme Q was obtained.

EXAMPLE 5 Candida curvata FERM P-632 was cultured in the same way as in Example 4, and 68 g dried cells were obtained. One gram of the dried cells was found to contain 32 pg coenzyme Q10, and 15.0 mg pure coenzyme Q was obtained.

EXAMPLE 6 The strains as indicated in the following Table 2 were cultured as in Example 4, and the results listed in Table 2 were obtained.

TABLE 2 Living Coencell zyme O Cnpaste in dried enzyme Strain used as dried cell cells (1;) (as g) s) Candida marina FERM P-854 4.05 480 15.5 Candida japanir'a FERM P-662 3615 400 14.6 Candida hugoricns'is' FERM P-666 60 300 9.0 Tumlupsis ingeniosa FERM P-665 105 450 47 'l'arulopxix capsu/igene: FERM P-667 65 260 16.9 'l'arulupsix aeria FERM P-664 80 300 16,3

EXAMPLE 7 Trichosporon byrdii FERM P 851 was inoculated on a 5 1 medium containing 5 g/dl glucose, 0.5 g/dl malt extract, 0.5 g/dl yeast extract and 0.2 g/dl ammonium sulfate, of pH 5.5, and cultured at 30C for 26 hours in the same way as in Example 4, and 74.5 g living cell paste (as dry matter) was obtained. The cells were found to contain 330 micrograms coenzyme Q per gram of dry cell solids.

The cells were worked up as in Example 4, and 17.1 mg pure crystalline coenzyme Q was obtained.

EXAMPLE 8 Alcaligenes faecalis FERM P-849 was inoculated on 5 liters of a medium containing 5 g /dl glucose, 1 g/dl polypeptone. 0.2 g/dl yeast extract and 0.5 g/dl meat extract. of pH 7.0, and cultured at 30C for 28 hours in the same way as in Example 4.

From the culture broth, 48.0 g living cells (as dry matter) were obtained, and the cells were found to contain 450 micrograms coenzyme Q per gram of dried cells.

The cells were treated in the same way as in Example 1, and 15.4 mg pure coenzyme Qw was obtained.

EXAMPLE 9 The yeasts indicated in Table 3 were cultured in the same way as in Example 4, and the living cells obtained (as dried cells), the coenzyme O content of the cells, and the amount of pure crystalline coenzyme Q obtained are listed in the following Table 3.

What we claim is: l. A method of producing coenzyme Qm which comprises culturing a microorganism selected from the group consisting of Rhodotorula flava [F0 1193, Rhodotorula glulinis 1E0 667, Rhadatorula rubra FERM P-632, Rhodotarula peneaus 1E0 930,

Rhodotorula pallida 1E0 715, Rhodotorula marina lFO 879, Rhodotorula texensis IFO 932, Cryptocaccus neoformans FERM P-633, Cryptocaccus albidus 1E0 610,

Cryptococcus laurentii [F0 609, Cryptococcus terreus 1E0 727, Cryptococcus luteolus lFO 61 l Sporobolomyces roseus lFO 1031, Sporobolomyces pararoseus [F0 1 103, Sporobolomyces salmonicolor lFQ 374, Sporobolomyces gracilis FERM P-633, Candida curvata ATCC 10567, Candida curvata FERM P663, Candida japonica FERM P-662, Candida bogoriensis FERM P-666, Candida marina FERM P-854, Torulopsis ingeniasa FERM P-665, Torulopsis capsuligenes FERM P-667, Torulopsis aeria FERM P-664, Rhodasporidium .rphaerocarpum F ERM P-856, Trichosporon byrdii FERM P-851, Trichasporon cutaneum FERM P-850, Aureobasidium pullulans FERM P-852, Bullera alha FERM P-855, and Rhodosporidium dibovatum FERM P-857 on a nutrient medium containing sources of assimilable carbon and nitrogen, inorganic salts, and organic nutrients until intracellular coenzyme 0, is produced in said microorganism.

2. A method as set forth in claim 1, wherein said coenzyme Q10 is recovered.

3. A method as set forth in claim 1, wherein said coenzyme Q is recovered in the form of crystals. 

2. A method as set forth in claim 1, wherein said coenzyme Q10 is recovered.
 3. A method as set forth in claim 1, wherein said coenzyme Q10 is recovered in the form of crystals. 